JPH01254868A - Method of measuring antigen material with extra-high sensitivity - Google Patents

Abstract

PURPOSE:To enable measurement with high sensitivity by forming a complex constituted of an antigen material to be measured of a liquid to be inspected and an active component, then conjugating the complex with a carrier and dissociating the complex from the simple substance after cleaning, then conjugating the same further with another carrier. CONSTITUTION:After the complex constituted of the antigen material to be measured of the liquid to be inspected and the active component is formed, the complex is conjugated with the carrier. The complex is otherwise formed on the carrier. The carrier conjugated with the complex is cleaned and thereafter, the complex is dissociated from the carrier. After this complex is conjugated with another carrier, the carrier is cleaned and the complex on the carrier is measured. The active component referred to is either the monoclonal antibody or polyconal antibody obtd. by immunizing an antigen to be measured or a material having the same antigen recognition part as the antigen or the antibodies and the component which generates an antigen-antibody reaction with these antibodies.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to an ultrasensitive method for measuring antigenic substances. [Prior Art] The measurement of antigenic substances in biological components, especially human body fluids, is clinically very difficult. is important. Measurement of antigens is widely used for endocrine testing by measuring hormones, cancer diagnosis, and diagnosis of ambiguity disease.

Conventionally, the above-mentioned trace amounts of antigenic substances have been measured by immunoassay. In recent years, methods using carriers have been widely used in immunoassay methods. ELISA
There are competitive assay methods such as sandwich assay method, IRMA method, and second antibody solid phase assay method.

The conventional sandwich-type antigen measurement method traps the antigen in the test liquid on a carrier that does not contain antibodies.
There is a method of measuring this using a labeled antibody against the antigen.

[Problem to be solved by the invention]

In any of these measurement methods, measurement errors are restricted by background originating from label components nonspecifically bound to the carrier, and there is a limit to highly sensitive measurements.

The purpose of the present inventors is to provide a measurement method with unprecedented high sensitivity under such circumstances.

[Means for resolving issues]

The present invention comprises the following steps (A), (B), (C) and (D): Step (A) Secondary fal or (bl step).

(A) A step of forming a complex between the antigenic substance to be measured in the test liquid and the active ingredient, and then binding this complex to a carrier.

(bl Step of forming the complex on a carrier.

Step (B): A step of washing the carrier bound to the complex and then dissociating the complex from the carrier.

Step (C): A step of washing this carrier after binding this complex to another carrier.

Step (D); A step of measuring the complex on the carrier described in (C).

This is a method for measuring an antigenic substance, which is characterized in that it includes the following.

The present invention will be explained below in accordance with the order of the steps.

Regarding step (A), the test liquid may be, for example, blood? i? , blood samples, body fluids such as cerebrospinal fluid, saliva, and urine, and buffer solutions. Antigenic substances to be measured include all substances that have an antigenic site as an antigen;
Virtually all substances that can be measured by conventional immunoassays can be measured. Examples include enzymes such as γ-glutamyl transpebudidase (γ-CTP), alkaline phosphatase, and glycosyltransferase, and thyroid stimulating hormone (
Protein hormones such as TSH), luteinizing hormone (LH), placental gonadotropin (hCG), insulin, secretin, growth hormone (GH), fibrin degradation product (FDP), and C-reactive protein (CRP) ), α, −
Acidic glycoprotein (α.

-AGP), α, -antitrypsin (α, -AT)
, α1-plasmin inhibitor (αz-P■), β-ohmicroglobulin (βt-y, ).

Plasma proteins such as immunoglobulin, oncofetal proteins such as α-fetoprotein (AFP), carcinoembryonic protein (CEA), and fetal ferritin, cells such as lymphocytes, viruses and microorganisms, cell surface antigens, etc., digoxin , thyroxine, triiodotyrosine, cortisol, and prostaglandins. These antigens can be measured in the test solution not only in a free state (they can also be measured in a state bound to immune complexes or binding proteins).

The active ingredient is 1 or 1 and 2 below.

1. Antibodies. The antibody referred to herein may be either a monoclonal antibody or a polyclonal antibody obtained by immunization with an antigen to be measured or a substance having the same antigen recognition site as the antigen by a known method.

2. A component that causes an antigen-antibody reaction with the above antibody.

(e.g. antigen, anti-antibody, etc.) These active ingredients are usually used in step (A) or/and step (
One or more functional groups involved in the bonding of the complex and the carrier in C) may be combined and used.

Here, the functional group includes, for example, habten such as dinitrophenyl group or trinitrophenyl group, biotin, -
Examples include antibodies or antigens other than the antigen to be measured and the corresponding antibody bound via a 5-S-bond, the above-mentioned Habten or the above-mentioned biotin, and the like.

The functional group is preferably one that is not inhibited from binding to the carrier by the components in the test solution in step (A), is difficult to detach from during washing in step (B), and facilitates dissociation during dissociation. , process (C)
The functional group involved in the bonding is preferably a functional group that can efficiently bond to another carrier from the solution dissociated in the dissociation step (B).

Also, at least one of these active ingredients is present in step (D).
to which the label used for measurement is attached.

The label may be any substance used in immunoassays, including enzymes, radioactive substances, luminescent substances, fluorescent substances, metal compounds, and the like.

For example, enzymes include peroxidase, β-D-galactosidase, and alkaline phosphatase, radioactive substances include iodine and hydrogen, fluorescent substances include fluorescein isothiocyanate, and luminescent substances include acridium salts.

These functional groups and labels may be bound to the active ingredient via a carrier that does not affect the steps (A) to (D), especially when the active ingredient is a low molecular weight. Preferred carriers include, for example, non-specific rabbit IgG, bovine serum albumin, dextran, and the like.

As a method for binding a label, any binding method for binding a label to an antibody or an antigen in conventional immunoassays may be used.

Formation of a complex composed of an antigen and an active ingredient is carried out by adding 111 or two or more active ingredients to a test liquid. 2
It is preferable to use more than one type of active ingredient and to use the active ingredient that binds a functional group and the active ingredient that binds 186 separately.

Formation of the complex is performed under conditions commonly used for antigen-antibody reactions. It is generally formed at 0 to 45°C for several hours to several 10 hours, preferably at 20 to 37°C for 1 to 6 hours. The complex thus formed is bound to a carrier.

Examples of the carrier include all the carriers used in conventional immunoassay methods, such as polystyrene, polyacrylic, Teflon, glass, agarose, and the like. Moreover, the shape may be any shape.

The carrier must have a reactive group for binding the complex formed in step (A) or for forming a complex on the carrier.

The reactive group that binds to the carrier is the antigen to be measured in the complex,
Active ingredients, functional groups, 4! ! Any substance that binds to the immunologically active site newly created by complex formation can be used as a reactive group.

The reactive group is preferably a reactive group that can easily dissociate the complex in step (B).

Examples of such reactive groups include common ones corresponding to functional groups, such as: 1) When the functional group is a dinitrophenyl group, trinitrophenyl group, etc., antibodies against these groups may be used.

2) When the functional group is biotinylated, examples include avidin or streptavidin.

3) When the functional group is an antigen or an antibody via a -5-S- bond, examples include the corresponding antibody or antigen.

Binding of the reactive group to the carrier is carried out by a known method for preparing carriers in immunoassays.

For binding of the complex to the carrier, conditions commonly used in the above-mentioned immunoassay using a carrier are employed.

It is desirable to add the active ingredient and the carrier to the test solution at the same time and form a complex on the carrier because this can simplify the process.

For washing, conditions commonly used for immunoassays using carriers are employed.

The dissociation of the complex is preferably carried out without decomposing the complex. When the binding between the complex and the carrier is due to an antigen-antibody reaction, the complex can be dissociated with acid, alkali, high concentration inorganic salt, etc. by making the binding constant of the antigen-antibody reaction smaller than the binding constant of complex formation. I can do it.

A more preferable method for dissociating the complex from the carrier without decomposing it is to add a substance having the same reactive site as the functional group involved in bonding the complex and the carrier.

For example, when the functional group is dinitrophenyl, it is a dinitrophenyl amino acid (e.g. dinitrophenyl lysine), when the functional group is biotin, it is biotin, and when it is an antigen, antibody, habuten, or biotin bound via -5-S-, etc. is -5
A reagent that cleaves -3- is used.

2■yu standing chant 2 public works As the carrier, those listed in step (A) are used.

The reactive group that binds to the carrier is the antigen to be measured in the complex,
Any active ingredient, functional group, label, or anything that binds to the newly created immunologically active site by complex formation can be used as a reactive group.

In addition to the reactive groups shown in step (A), preferred reactive groups include components to be measured in the complex, active components, labels, or
Examples include reactive groups that bind to newly generated immunologically active sites due to complex formation.

A reactive group that binds to the component to be measured in the complex, i.e., the active ingredient, through an antigen-antibody reaction is particularly preferred, since the introduction of one functional group can be omitted.

When using the same reactive group as that used in step (A), it is necessary to separate the complex from the solution rinsed in step (B). In order to avoid this, it is preferable to use a different reactive group from that in step (A). In this case, the step of dissociating (B) and the step of bonding to the carrier (C) can be performed simultaneously.

For binding of the complex to the carrier, as in step (A>), conditions commonly used in the immunoassay using a carrier are employed.

The above steps CB) and (C) may be repeated as necessary.

In order to measure the complex on the carrier, there is a method of measuring the label introduced into the active ingredient described in step (A).

As explained above, the present invention can measure antigenic substances that can be measured by conventional immunoassay methods with higher sensitivity than conventional methods.

When performing antigen measurement, it is possible to use a combination of complexes depending on the purpose as described below.

1. In sandwich-type measurements, antibodies obtained from two types of animals that can be sandwiched are used as active ingredients. A complex is formed by bonding a functional group to one side and a label to the other side, and in step (A), the complex is bonded to a carrier by the functional group,
By using a carrier bound with an anti-antibody against the antibody bound with a functional group in step (C), non-specific binding of the antibody bound with a label to the carrier is reduced, and in step (D), the complex on the carrier is reduced. Measure the label inside.

2゜When using two types of antibodies obtained from animals of the same species as active ingredients, one is bound with two types of functional groups and the other with a label to form a complex, and in step (A) the functional groups are bonded. By using a carrier that can be bound to a carrier and bound to the other functional group in step (C), non-specific binding of the label-bound antibody to the carrier can be reduced, and in step (D) the antibody on the carrier can be bound to the carrier. Measure the label of the complex.

3. In the case of competitive assays, antigens and antibodies are used as active ingredients. A label is bound to the antigen, a functional group is bound to the antibody, and a complex is formed. In step (A), the functional group is bound to a carrier, and in step (C), the carrier bound with the anti-antibody against the antibody is By using this method, nonspecific binding of the label-bound antigen to the carrier is reduced, and in step (D), the label in the complex on the carrier is measured.

The present invention will be explained below with reference to Examples, but the present invention is not limited thereto.

〔Example〕

Example-1 0.01 containing 0, IM NaCl, 1mM MgCl, 0.1% bovine serum albumin and 0.1% NaN5
? A sodium phosphate buffer, phi 7.0, was prepared and designated as Buffer A.

A standard product of the N Tanuki U semi-nihili hTslI kit "Daiichi" (Daiichi Radioisotope, Tokyo) was used.

1 G, F (ab')
(ab')t is converted into Fab' by pepsin digestion of IgG.
are obtained by reduction of F(ab')t using known methods (
Ishikawa et al., Journal of Immunoassay (J, I
mmunoassay) Volume 4, Page 209 (1983
)).

■Mercaptosuccinyl-monoclonal mouse (
Anti-hTSHβ-subunit) IgG+ tIi monoclonal mouse (anti-hTsIlβ-subunit) IgG+, (7 Lin Croft St. Louis X, a:
X+)-state) to S-acedermercaptosuccinic
A thiol group was introduced by a known method using anhydride [Ishikawa et al., Journal of Immunoassay (cited above)]. The number of thiol groups introduced per molecule was 10.5.

2. Preparation of maleimido-dinitrophenyl-L-lysine 5.5mM dinitrophenyl-L-lysine, 5mjl
0.1 M sodium phosphate buffer containing EDTA, PH
7,0,0, hl and 5mM N-succinimidyl-6-
0.10 ml of N,N-dimethylformamide containing maleimidohexanoate (described above) was reacted at 30° C. for 30 minutes.

3. Dinitrophenyl monoclonal mouse (anti-hT
sHβ-subunit ) IgG, of! II mercaptosuccinyl monoclonal mouse (anti-hTsHβ-
subunit) 5mM in which Igclo, t■ was dissolved
0.1 sodium phosphate buffer containing EDTA, pH
6,0,4,5ml and maleimide-dinitrophenyl-
A reaction solution obtained by reacting 0.5 ml of L-lysine solution at 30° C. for 30 minutes was gel-filtered through a Sephadex G-25 column. The column size is 1. OX30cm+, eluent is O, LM sodium phosphate solution II, pl+ 7.
0 was used. Monoclonal mouse (anti-hTsIIβ
-subunit)! g, the number of dinitrophenyl groups introduced per molecule was 7.2.

Dinitrophenyl-Bovine Blood Albumin Characteristics 1 Prepared in a manner similar to that described above using bovine serum albumin (Fraction V, Armor Company, Kankakee, IL).

The number of dinitrophenyl groups introduced per molecule of bovine serum albumin was 7.0.

White - Sepharose 4B 8. '' hCG (2,5a+g), dinitrophenyl-bovine serum albumin ('Long) and non-specific mouse IgG
(20■) is CNBr according to the Pharmacia manual.
- Insolubilized in activated Sepharose 4B (Ig).

■G affinity +1 Rabbit (anti-hCG) prepared from rabbit (anti-hCG) IgG (Dakobasots, Glostrahup, Denmark)
F(ab')z, rabbit (anti-dinitrophenyl maggot serum albumin) IgG (Miles, Inc., Hercult, IN) and rabbit (anti-mouse IgG) IgG (
Medical and Biological Research Institute 1 Nagoya) are hcc, dinitrophenyl-bovine serum albumin, and non-specific mouse Ig, respectively.
Using a G-insolubilized Sepharose 4B column, pl+ 2.
5 [Kono et al., Journal of
Biochemistry (J, Biochem,) No. 100
vol., p. 1247 (1986)).

β-D-galactosidase activity measurement β-D-galactosidase activity is 4-methylumbelliferyl β-
D-galactoside was reacted as H at 30°C for 16 hours, and then measured fluorescently using a known method.
in, Biochem,) Volume 21, Page 310 (19
84) ), the fluorescence intensity was measured with 0,1M glycine-Na01 in which 10-''M4-methylumbelliferone was dissolved.
Measurements were made using 1J fat sealed liquid, pHlO, 3 as a standard.

Affinity-purified rabbit anti-(hCG) tab' was N.

By a known method using N'-o-phenylene dimaleimide as a crosslinking agent [Ishikawa et al., Scandinavian Journal of Immunology (Scand, J., Immunol.
) Volume 8 (Supplement 7) Page 43 (197B) ) Labeled with β-D-galactosidase. β-D-galactosidase 1
2.1 Fab' were bound to the molecule.

0.5 ml of Buffer A containing 0.8 ml of Fab'-β-D-galactodase was passed through a column of non-specific mouse serum protein-Sepharose 4B using Buffer A. The amount of labeled antibody was calculated from the enzyme activity.

7Finity main n rabbit (anti-dinitrophenyl-bovine serum albumin) TgG solution (0.1 g/l) or affinity purified rabbit (anti-mouse IgG) rgG
solution (0.1 g/l) onto the surface of a polystyrene ball [diameter 3.2 wm (Precision Plastic Ball Co., Chicago)] in a known manner [Ishikawa et al., Scandinavian Journal of Immunology, vol. out)]
Insolubilized by physical adsorption.

Nijidan 9 Harada Affinity Purified Rabbit Anti (hCG) Fab″-β
-Buffer A0 containing D-galactosidase 200fo+
．． 0.01ml of buffer solution A0.01ml containing 0.1mg of non-specific mouse IgG was added to 0.01ml and left at 20°C for 2 hours.

Separately, dinitrophenyl-monoclonal mice (anti-hT
slIβ-subunit) IgG+, 150 fmol
Buffer including? &A0.02ml non-specific rabbit F(ab
0.02ml of buffer A containing 0.1a+g of ')x was added and left at 20°C for 2 hours.

The two solutions were then mixed and reacted overnight at 20°C with 0.09 ml of buffer A containing various concentrations of hTsH standards. Two polystyrene balls were added and reacted at 20°C for 4 hours.

After removing the reaction solution, place the polystyrene ball in buffer solution A2+.
Washed twice with 1.

Buffer A0.15 containing 1mM dinitrophenyl-lysine, non-specific rabbit F(ab')gO, Lmg
m1 and affinity purified rabbit (anti-mouse fgG)
Add 2 RGC insolubilized polystyrene balls and heat at 20°C.
, and reacted for 4 hours.

Affinity purified rabbit (anti-mouse IgG) IgG
After washing the insolubilized polystyrene balls in the same manner as above, the activity of β-0-galactosidase bound to the polystyrene balls was measured after reaction at 30° C. for 16 hours.

The results are shown in FIG. 1 together with Comparative Example-1, which will be described later. That is, FIG. −
It is a graph shown in relation to the fluorescence intensity (vertical axis) corresponding to galactosidase.

Comparative Example-1 Buffer, hTSH standard, β-D-galactosigase activity measurement, affinity purification rabbit (anti-hcG) Fa
Preparation of b'-β-D-galactosidase, monoclonal mouse (anti-hTSHβ-subunit/)) IgG
.

The insolubilized polystyrene balls were prepared according to Example-1 ◆ U-jump Ω limp monoclonal mouse anti-(hTSHβ-subunit)
TgG, insolubilized polystyrene balls were reacted with 0.15+nl of buffer A containing standard hTSH at 20°C overnight.

After removing the reaction solution and washing the polystyrene ball twice with 2 ml of buffer A, the affinity-purified rabbit (anti-h
CG) Fab'-β-D-galactosidase 200
fmol and non-specific rabbit F(ab')go, 1 mg
The mixture was reacted with 0.15 ml of buffer A containing the following at 20°C for 4 hours.

After washing the polystyrene balls at the same time as above, the β-D-galactosidase activity bound to the polystyrene balls was removed.
Measurement was carried out after reaction at 0°C for 1 hour. The results are shown in Figure 1.

Example-2 Preparation of TgG, F(ab')z+Fab', protein-Sepharose 4B steel, affinity purification of IgG, affinity TFT! <Anti-dinitrophenyl-bovine serum albumin) IgG insolubilized solid phase, affinity purified rabbit (anti-mouse IgG) IgG insolubilized solid phase was prepared according to the method of Example-1.

A standard product of the Shisaki hGII RIA kit (Guinabot Tokyo) was used.

It was carried out according to the method of Example-1. The number of dinitrophenyl groups introduced per monoclonal mouse (anti-hGH) IgG+ molecule was 5.7.

The method of Example-1 was followed. 4.1 Fab' were bound to one molecule of β-D-galactosidase.

Buffer A0.0 ha+ containing 200 rmol of Fab'-β-D-galactosidase and 1 mg of non-specific mouse IgG O
1 ml was added and left at 20°C for 2 hours.

Separately, dinitrophenyl monoclonal mice (anti-hG
11) Buffer A0.02 containing IgD+150 fmol
0.02 ml of buffer A containing 0.1 mg of non-specific rabbit F(ab')t was added to ml and left at 20°C for 2 hours.

The two solutions were then mixed to obtain different concentrations of hGl! 41
! ! Affinity purified rabbit (
Anti-dinitrophenyl-bovine serum albumin) 2 gG insolubilized polystyrene poles were added and reacted at 20°C for 4 hours.

After removing the reaction solution, place the polystyrene pole in buffer solution A2.
Washed twice with all.

Buffer A0.15n containing 1+mM dinitrophenyl-cy-lysine, 0.1 mg of non-specific rabbit F(ab')g
+1 and affinity purified rabbit (anti-mouse [gG)
Add 2 IgG insolubilized polystyrene balls and heat at 20°C.
, and reacted for 4 hours.

Affinity purified rabbit (anti-mouse IgG) IgG
The insolubilized polystyrene balls were washed in the same manner as described above, and the activity of β-D-galactosidase bound to the polystyrene balls was measured after reaction at 30° C. for 16 hours. The results are shown in FIG. 2 together with Comparative Example-2 described later.

That is, FIG. 2 shows the results of the present invention and the conventional example with hG
It is a graph showing the relationship between Hff, degree C9g/tube (horizontal axis) and fluorescence intensity (vertical axis) corresponding to specifically bound β-D-galactosidase.

Comparative Example-2 Buffer, β-D-galactosidase activity measurement, monoclonal mouse (anti-hGIl) rgc insolubilized solid phase!
The product manufactured by II was in accordance with Example-1.

hGI+ standard product, purified rabbit (anti-hGH) Fab'-β
-D-galactosidase! The method of Example 2 was followed.Two plates per mouse were incubated with buffer A0.15-1 containing standard hG)I, monoclonal mouse (anti-hGH) rgG, and insolubilized polystyrene balls at 20°C overnight. I let it happen.

After removing the reaction solution, the polystyrene pole was washed twice with 2 ml of buffer A, and rabbit (anti-fart II) Fab'
- Buffer A0.15 containing 200 fmol of β-D-galactosidase and non-specific rabbit F(ab')z O,IB
It was reacted with m1 at 20°C for 4 hours.

The polystyrene poles were washed in the same manner as above to remove β-D-galactosidase activity bound to the polystyrene poles.
Measurement was carried out after reaction at 0°C for 1 hour.

The results are shown in Figure 2.

[Effect of the invention 5 As explained above, the method of the present invention can measure antigens with ultra-high sensitivity, so it contributes to the diagnosis of various hormone secretion abnormalities such as thyroid diseases and hypopituitary gland disorders, and various N-induced infections. It is something to do.

[Brief explanation of the drawing]

FIGS. 1 and 2 are graphs showing the β-D-galactosidase activities of the present invention and conventional examples.

Claims (1)

[Claims] 1. A method for measuring an antigenic substance characterized by including the following steps (A), (B), (C) and (D) Step (A): The following (a) or (b) Process. (a) A step of forming a complex composed of the antigenic substance to be measured in the test liquid and the active ingredient, and then binding this complex to a carrier. (b) Forming the complex on a carrier. Step (B): A step of washing the carrier bound to the complex and then dissociating the complex from the carrier. Step (C): A step of washing this carrier after binding this complex to another carrier. Step (D): A step of measuring the complex on the carrier described in (C).